The long term objective of our laboratory is to understand the regulation of steroid hormone production and the molecular bases of congenital disorders in the steroidogenic enzymes. To this end we have already cloned cDNA and genomic DNA for three adrenal cytochrome P450 enzymes involved in steroid production, the cholesterol side-chain cleavage enzyme (P450scc), 21-hydroxylase (P450c21) and 17Alpha-hydroxylase/17,20 lyase (P450c17). We have used these to study regulation of the cholesterol side-chain cleavage enzyme and 17Alpha-hydroxylase/17,20 lyase and genetic lesions in cholesterol side-chain cleavage and 21-hydroxylase deficiency. However, none of the presently available technology has succeeded in isolating, characterizing or cloning proteins, cDNAs or genes for the key enzymes 3Beta-hydroxysteroid dehydrogenase (3BetaHSD) and delta4, delta5 isomerase (Isom). While deficiency of 3BetaHSD has generally been regarded as rare, recent data indicate that mild enzymatic defects in 3BetaHSD and/or Isom are a common cause of hirsutism, virilism, polycystic ovarian disease, and infertility in both sexes. The regulation of 3BetaHSD/Isom is also of great interest as their activity is low in the fetal adrenal and induced at birth; by contrast, adrenal carcinomas are often characterized by deficient 3BetaHSD/Isom activity. To obtain cDNA probes to study the structure, regulated expression and disease-causing mutations in these key enzymes we propose a radical new approach to biological identification of their cDNA clones. An enriched source of mRNA will be used to clone cDNA in an established yeast vector known to permit expression and membrane insertion of mammalian NAD-requiring enzymes. Yeast recombinants will be screened for expression of 3BetaHSD or Isom by appropriate conversion of 3H-labeled steroidal substrates and assay of the steroidal products by high pressure liquid chromatography. This proposed system will first be tested and optimized using another steroidogenic enzyme cDNA (P450c17) recently cloned in our lab.